During the previous funding period, we have isolated stable forms of thrombin-activated and fXa-activated human and porcine fVIII and have used these preparations to study the assembly of the intrinsic fXase complex (fIXa/fVIIIafX/phospholipid) by using kinetic and fluorescence binding methods. We have described a novel anticoagulant property of heparin: antithrombin-independent inhibition of intrinsic fXase. We have studied the functional role of the fVIIIa A2 subunit and the mechanism of inhibition of fVIIIa by clinically important inhibitory anti-A2 antibodies. We have mapped the A2 epitope to a 25 residue segment by using a homolog scanning mutagenesis method employing active, recombinant hybrid human/porcine fVIII molecules. We have participated in a collaborative project with Wolfram Bode that has produced a 2.8 A X-ray structure of full-length porcine D-FPR-fIXa. During the next project period, we will map inhibitory epitopes in the fVIII light chain. We will characterize inhibitory fVIII epitopes using alanine scanning mutagenesis. We will also use hybrid human/porcine fVIII molecules to determine the structural basis for the decreased dissociation rate constant of the porcine A2 subunit and increased coagulant activity of porcine fVIII compared to human fVIII. We will use the X-ray structure of fIXa as guide for alanine scanning mutagenesis studies to characterize the fIXa binding site for heparin. We will also test the hypothesis that the extremely low catalytic activity of fIXa in the absence of cofactors is due to the presence of Glu219, which is unique in fIX and which in molecular dynamics simulations disorders the specificity pocket of fIXa. We will do this by making a Glu219->Gly fIlXa mutant and characterizing its enzymatic properties. We will test the hypothesis that functionally important segmental flexibility exists between a module or modules in fIXa by studying decays in fluorescence anisotropy of dansyl-EGR-fIXa in the presence and absence of fVIIIa and PL. These studies may have a direct impact in the treatment of hemophilia and the development of novel antithrombotic drugs.